Composition and methods for cell killing

ABSTRACT

A method of generating a change in a cellular process of a target cell is disclosed. The method comprises contacting the target cell with a solid buffer, so as to alter an intracellular pH value in at least a portion of the cell, thereby generating the change in a cellular process of a target cell of a multicellular organism. The method may be used to kill either eukaryotic or prokaryotic cells. Pharmaceutical compositions and devices are also enclosed.

RELATED APPLICATION

This Application claims the benefit of U.S. Provisional PatentApplication No. 60/732,130 filed on Nov. 2, 2005, the contents of whichare hereby incorporated in its entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to compositions and methods for killingcells based on the titration thereof with solid buffers.

Various forms of cellular material are known to be harmful andpotentially lethal to man. For example, cancerous cells are the secondleading cause of death in the United States, after heart disease (Boringet al., C A Cancel J. Clin. 43:7 (1993)). Cellular microorganisms arealso responsible for a wide range of diseases. Cell killing and targetedcell killing (e.g., cancer) are highly investigated in the biotechnologyindustry.

A cancer is a malignant tumor of potentially unlimited growth. It isprimarily the pathogenic replication (a loss of normal regulatorycontrol) of various types of cells found in the human body. Initialtreatment of the disease is often surgery, radiation treatment or thecombination of these treatments, but locally recurrent and metastaticdisease is frequent. Chemotherapeutic treatments for some cancers areavailable but these seldom induce long term regression. Hence, they areoften not curative. Commonly, tumors and their metastases becomerefractory to chemotherapy, in an event known as the development ofmultidrug resistance. In many cases, tumors are inherently resistant tosome classes of chemotherapeutic agents. In addition, such treatmentsthreaten non-cancerous cells, are stressful to the human body, andproduce many side effects. Improved agents are that are capable oftargeting cancerous cells are therefore needed.

Microorganisms can invade the host tissues and proliferate, causingsevere disease symptoms. Pathogenic bacteria have been identified as aroot cause of a variety of debilitating or fatal diseases including, forexample, tuberculosis, cholera, whooping cough, plague, and the like. Totreat such severe infections, drugs such as antibiotics are administeredthat kill the infectious agent. However, pathogenic bacteria commonlydevelop resistance to antibiotics and improved agents are needed toprevent the spread of infections due to such microorganisms.

One of the principal concerns with respect to products that areintroduced into the body or provide a pathway into the body is bacterialinfection. Avoiding such infections with implantable medical devices canbe particularly problematic because bacteria can develop into biofilms,which protect the microbes from clearing by the subject's immune system.As these infections are difficult to treat with antibiotics, removal ofthe device is often necessitated, which is traumatic to the patient andincreases the medical cost. Accordingly, for such medical apparatuses,the art has long sought means and methods of rendering those medicalapparatuses and devices antibacterial and, hopefully, antimicrobial.

The general approach in the art has been that of coating the medicalapparatuses, or a surface thereof, with a bactericide. However, sincemost bactericides are partly water soluble, or at least requiresufficient solubilization for effective antibacterial action, simplecoatings of the bactericides have been proven unreliable. For thisreason, the art has sought to incorporate the bactericides into themedical apparatus or at least provide a stabilized coating thereon.

Alternatively, materials can be impregnated with antimicrobial agents,such as antibiotics, quarternary ammonium compounds, silver ions, oriodine, which are gradually released into the surrounding solution overtime and kill microorganisms there. Although these strategies have beenverified in aqueous solutions containing bacteria, they would not beexpected to be effective against airborne bacteria in the absence of aliquid medium; this is especially true for release-based materials,which are also liable to become impotent when the leaching antibacterialagent is exhausted.

Any agent used to impair biofilm formation in the medical environmentmust also be safe to the user. Certain biocidal agents, in quantitiessufficient to interfere with biofilms, also can damage host tissues.Antibiotics introduced into local tissue areas can induce the formationof resistant organisms which can then form biofilm communities whoseplanktonic microorganisms would likewise be resistant to the particularantibiotics. Any anti-biofilm or antifouling agent must furthermore notinterfere with the salubrious characteristics of a medical device.Certain materials are selected to have a particular type of operatormanipulability, softness, water-tightness, tensile strength orcompressive durability, characteristics that cannot be altered by anagent added for anti-microbial effects.

Food is also a source of bacterial infection and the preservationthereof is of utmost importance in order to keep food safe forconsumption and inhibit or prevent nutrient deterioration ororganoleptic changes, causing food to become less palatable and eventoxic. Preservation of food products can be achieved using a variety ofapproaches. Physical manipulations of food products that have apreservative effect include, for example, freezing, refrigerating,cooking, retorting, pasteurizing, drying, vacuum packing and sealing inan oxygen-free package. Some of these approaches can be part of a foodprocessing operation. Food processing steps preferably are selected tostrike a balance between obtaining a microbially-safe food product,while producing a food product with desirable qualities.

With the increasing use of polymeric materials for construction ofmedical apparatuses and packaging and handling of food products,utilizing an antimicrobial polymer has become ever more desirable.Although, antimicrobial polymers exist in the art, there is still a needfor an improved antimicrobial polymer coating that may be easily andcheaply applied to a substrate to provide an article which has excellentantimicrobial properties and which retains its antimicrobial propertiesin a permanent and non-leachable fashion when in contact with cellularmaterial for prolonged periods.

U.S. Pat Appl No. 20050271780 teaches a bactericidal polymer matrixbeing bound to an ion exchange material such as a quaternary ammoniumsalt for use in food preservation. This polymer matrix kills bacteria byvirtue of incorporating therein of a bactericidal agent (e.g. thequaternary ammonium salt). The positive charge of the agent merely aidsin electrostatic attraction between itself and the negatively chargedcell walls. In addition, the above described application does not teachuse of solid buffers having a buffering capacity throughout their entirebody.

U.S. Pat. Appl. No. 20050249695 teaches immobilization of antimicrobialmolecules such as quarternary ammonium or phosphonium salts (cationic,positively charged entities) covalently bound onto a solid surface torender the surface bactericidal. The polymers described herein areattached to a solid surface by virtue of amino groups attached theretoand as such the polymer is only capable of forming a monolayer on thesolid surface.

U.S. Pat. Appl. No. 20050003163 teaches substrates having antimicrobialand/or antistatic properties. Such properties are imparted by applying acoating or film formed from a cationically-charged polymer composition.

The activity of the polymers as described in U.S. Pat. Appl. Nos.20050271780, 20050249695 and 20050003163 relies on the direct contact ofthe bactericidal materials with the cellular membrane. The level oftoxicity is strongly dependent on the surface concentration of thebactericidal entities. This requirement presents a strong limitationsince the exposed cationic materials can be saturated very fast in ionexchange reactions.

In addition, none of the above described U.S. patent applications teachkilling eukaryotic cells. Nor do they teach the in vivo use of polymersas cytotoxic agents against either eukaryotic or prokaryotic cell types.Furthermore, none of the above mentioned U.S. patent applications teachconfiguration of the polymers to selectively kill certain cell types.

There thus remains a need for and it would be highly advantageous tohave agents capable of cytotoxic action both against eukaryotic andprokaryotic cells.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of generating a change in a cellular process of a target cell ofa multicellular organism, the method comprising contacting the targetcell with a solid buffer, so as to alter an intracellular pH value in atleast a portion of the cell, thereby generating the change in a cellularprocess of a target cell of a multicellular organism.

According to another aspect of the present invention there is provided amethod of killing a target cell of a multicellular organism comprisingcontacting the target cell with a solid buffer, so as to alter anintracellular pH value in at least a portion of the cell, therebykilling the target cell.

According to yet another aspect of the present invention there isprovided a method of generating a change in a cellular process of atarget cell, the method comprising contacting the target cell with asolid buffer, the solid buffer being anionic, so as to alter anintracellular pH value in at least a portion of the cell, therebygenerating the change in a cellular process of a target cell.

According to still another aspect of the present invention there isprovided a method of generating a change in a cellular process of atarget cell, the method comprising contacting the target cell with asolid buffer, wherein the solid buffer comprises a buffering layer and awater permeable layer being disposed on an external surface of thebuffering layer, so as to alter an intracellular pH value in at least aportion of the cell, thereby killing the cell.

According to an additional aspect of the present invention there isprovided a method of killing a cell comprising contacting the cell witha solid buffer, the solid buffer comprising a volumetric bufferingcapacity greater than 50 mM H⁺/l.pH, and a pH either greater than pH 8,or less than pH 4.5, thereby killing the cell.

According to still an additional aspect of the present invention thereis provided a method of selecting a solid buffer capable of killing acell, the method comprising selecting a solid buffer having a volumetricbuffering capacity greater than 50 mM H/l.pH, and a pH greater than pH 8or a pH less than pH 4.5, the solid buffer being capable of killing thecell.

According to still an additional aspect of the present invention thereis provided a method of killing a sub-population of cells of interest,the method comprising contacting a sample which comprises thesub-population of cells of interest with a solid buffer having avolumetric buffering capacity and a pH selected suitable forspecifically killing the sub-population of cells of interest, therebykilling the sub-population of cells of interest.

According to yet an additional aspect of the present invention there isprovided an article of manufacture comprising:

(i) a support; and

(ii) a solid buffer layer being attached to at least part of a surfaceof the support, the solid buffer comprises a buffering layer and an ionpermeable layer being disposed on an external surface of the bufferinglayer.

According to still an additional aspect of the present invention thereis provided an article of manufacture comprising:

(i) a support; and

(ii) a solid buffer layer being attached to at least part of a surfaceof the support, the solid buffer being anionic.

According to still an additional aspect of the present invention thereis provided a use of a solid buffer for the manufacture of a medicamentfor treating a medical condition associated with a pathological cellpopulation.

According to a further aspect of the present invention there is provideda pharmaceutical composition comprising as an active ingredient a solidbuffer and a pharmaceutically acceptable carrier or diluent.

According to yet a further aspect of the present invention there isprovided an assay for selecting an optimal solid buffer for killing acell of interest, the assay comprising:

(i) contacting a plurality of cells with a plurality of solid bufferagents;

(ii) identifying a solid buffer agent of the plurality of solid bufferagents capable of killing a cell of the plurality of cells, the solidbuffer agent being optimized for killing the cell of interest.

According to yet a further aspect of the present invention there isprovided a method of treating a medical condition associated with apathological cell population, the method comprising administering into asubject in need thereof a therapeutically effective amount of a solidbuffer so as to alter at least a portion of an intracellular pH value ofthe pathological cell population, thereby treating the medical conditionassociated with the pathological cell population.

According to further features in preferred embodiments of the inventiondescribed below, generating the change results in death of the cell.

According to still further features in the described preferredembodiments the multicellular organism is a higher plant.

According to still further features in the described preferredembodiments the multicellular organism is a mammal.

According to still further features in the described preferredembodiments the contacting is effected in vivo.

According to still further features in the described preferredembodiments the contacting is effected ex vivo.

According to still further features in the described preferredembodiments the contacting is effected in vitro.

According to still further features in the described preferredembodiments the solid buffer comprises a pH gradient along at least aportion thereof.

According to still further features in the described preferredembodiments the solid buffer is internalized by the target cell.

According to still further features in the described preferredembodiments the solid buffer is attached to an affinity moiety.

According to still further features in the described preferredembodiments the affinity moiety is selected from the group consisting ofan antibody, a receptor ligand and a carbohydrate.

According to still further features in the described preferredembodiments the solid buffer is at least partially covered by aselective barrier.

According to still further features in the described preferredembodiments the selective barrier is a mechanical barrier.

According to still further features in the described preferredembodiments the solid buffer comprises a buffering layer and a waterpermeable layer being disposed on an external surface of the bufferinglayer.

According to still further features in the described preferredembodiments the water permeable layer is an open pore polymer.

According to still further features in the described preferredembodiments the open pore polymer is selected from the group consistingof PVOH, cellulose and polyurethane.

According to still further features in the described preferredembodiments the solid buffer is formulated in particles.

According to still further features in the described preferredembodiments the solid buffer is formulated as a spray.

According to still further features in the described preferredembodiments the solid buffer is encapsulated within the particles.

According to still further features in the described preferredembodiments the solid buffer is attached on the particle surface.

According to still further features in the described preferredembodiments the particles are selected from the group consisting ofpolymeric particles, microcapsules liposomes, microspheres,microemulsions, nanoparticles, nanocapsules and nanospheres.

According to still further features in the described preferredembodiments the solid buffer is an anionic, ion exchange materialincorporated in a water permeable polymer matrix.

According to still further features in the described preferredembodiments the solid buffer is a cationic, ion exchange materialincorporated in a water permeable polymer matrix.

According to still further features in the described preferredembodiments the solid buffer comprises a cationic ion exchange materialand an anionic ion exchange material incorporated in a water permeablepolymer matrix.

According to still further features in the described preferredembodiments the cationic ion exchange material is selected from thegroup consisting of sulfonic acid and derivatives thereof, phosphonicacid and derivatives thereof, carboxylic acid and derivatives thereof,phosphinic acid and derivatives thereof, phenols and derivativesthereof, arsonic acid and derivatives thereof and selenic acid andderivatives thereof.

According to still further features in the described preferredembodiments the anionic ion exchange material is selected from the groupconsisting of a quaternary amine, a tertiary amine, a secondary amineand a primary amine.

According to still further features in the described preferredembodiments the solid buffer is a polymer.

According to still further features in the described preferredembodiments the solid buffer comprises an intrinsically ion conductingmatrix.

According to still further features in the described preferredembodiments the solid buffer is an ionomer.

According to still further features in the described preferredembodiments the ionomer is sulfonated tertafluorethylene copolymer(Nafion) and derivatives thereof.

According to still further features in the described preferredembodiments the solid buffer comprises a volumetric buffering capacitybetween about 20-100 mMH⁺/l pH.

According to still further features in the described preferredembodiments the solid buffer comprises a pH greater than pH 8.

According to still further features in the described preferredembodiments the solid buffer comprises a pH less than pH 4.5.

According to still further features in the described preferredembodiments the cell is a diseased cell.

According to still further features in the described preferredembodiments the solid buffer is attached to at least part of a surfaceof a support.

According to still further features in the described preferredembodiments the sample comprises at least a second sub-population ofcells, wherein the sub-population of cells of interest and the secondsub-population of cells exhibit different plasma buffering capacities.

According to still further features in the described preferredembodiments the treating is effected ex-vivo.

According to still further features in the described preferredembodiments the treating is effected in-vivo.

According to still further features in the described preferredembodiments the article of manufacture forms at least a part of apackaging material, a medical device, a fabric, a scaffold, a filter, ora bactericidal device.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing novel methods of affecting acellular change by contacting cells with a solid buffer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in colorphotograph. Copies of this patent with color photograph(s) will beprovided by the Patent and Trademark Office upon request and payment ofnecessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1A-B are graphs illustrating the spatial distribution of thenative and urea denatured forms of the protein phycocyanin in stripscontaining polyacrylamide based gels having a pH gradient. The graph ofFIG. 1A represents the results of the scan for native (non-denatured)phycocyanin. The graph of FIG. 1B represents the results of the scan for8M Urea denatured phycocyanin. The vertical axes represent theAbsorbance in O.D. units and the horizontal axes represent the positionon the scanned gel strip expressed in pH units.

FIGS. 2A-B are graphs illustrating the spatial distribution of thenative and urea denatured forms of the protein myoglobin in stripscontaining polyacrylamide based gels having a pH gradient. The graph ofFIG. 2A represents the results of the scan for native (non-denatured)myoglobin. The graph of FIG. 2B represents the results of the scan for8M Urea denatured myoglobin. The vertical axes represent the Absorbancein O.D. units and the horizontal axes represent the position on thescanned gel strip expressed in pH units.

FIGS. 3A-B are photographs representing two different stages of theresults of an experiment demonstrating pH dependent separation andredistribution of the two different proteins myoglobin and phycocyanin.FIG. 3A is a top view of the experimental chamber immediately after themixture of myoglobin and phycocyanin was placed in the middlecompartment 2. FIG. 3B is a top view of the same experimental chamberphotographed seven days following disposition of the mixture ofmyoglobin and phycocyanin in the middle compartment 2.

FIGS. 4A-C are composite photomicrographs illustrating the temporalvariation of GFP distribution in a cell following attachment of the cellto a pH modifying bead. FIG. 4A represents a cell (8) attached to a bead(6) at time zero (defined as the time of attachment of the cell to thebead). FIG. 4B represents the cell (8) attached to the bead (6), asphotographed ten minutes after the leftmost photograph was taken. FIG.4C represents the cell (8) attached to the bead (6), as photographedthirty (30) minutes after the leftmost photograph was taken. Thefluorescing point labeled by the thick white arrows, represents thefluorescence of GFP that migrated and accumulated at the point ofcontact between the bead 6 and the cell 8.

FIG. 5 is a graph illustrating the spatial distribution of Yellowfluorescent protein (YFP) on strips of immobiline containingpolyacrylamide based gels having a pH gradient. The vertical axisrepresents optical density, and the horizontal axis represents theposition along the IPG strip expressed in pH units.

FIGS. 6A-B are photomicrographs illustrating the cytotoxic effect ofNafion film on Jurkat cells. FIG. 6A illustrates Jurkat cells on anon-Nafion surface. FIG. 6B illustrates Jurkat cells on a Nafionsurface.

FIG. 7 is a line graph showing the percent of dead (red) Jurkat cellsfollowing exposure to the MVC/HT/56 A, B, C and D films of the presentinvention.

FIGS. 8A-D are photomicrographs illustrating the cytotoxic effect ofBIOACT 13, 15, 16 and 110 films on Jurkat cells using LIVE/DEAD®BacLight™ Bacterial Viability Kit (Molecular probes) in which dead cellsappear red and live cells appear green under a fluorescent microscope.FIG. 8A illustrates control Jurkat cells (with no exposure to bioactivefilm) after 1 minute. FIG. 8B illustrates Jurkat cells with BIOACT 13film added after 1 minute. FIG. 8C illustrates control Jurkat cells(with no exposure to bioactive film) after 10 minutes. FIG. 8Dillustrates Jurkat cells with BIOACT 13 film added after 10 minutes.

FIGS. 9A-C are photographs illustrating the anti-necrotic effect of ionexchange resin beads. FIG. 9A is a photograph of a necrotic tissue priorto the administration of the ion exchange resin beads. FIG. 9B is aphotograph of the same tissue following a two day application of the ionexchange beads. FIG. 9C is a photograph of the fabric to which the ionexchange beads were applied.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods of affecting cellular processesusing solid buffers. Specifically, the present invention may beexploited for a myriad of applications ranging from the killing ofdiseased cells in the body such as cancerous cells, to the killing ofharmful prokaryotic cells in the environment.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

This invention is the result of a serendipitous and unexpected findingby the present inventors. They demonstrated that biomolecules (e.g.proteins) typically comprise a pH characteristic which determines theirspatial distribution along a pH gradient (See Example 1). Furtherexperimentation provided evidence that this redistribution can alsooccur across a biological membrane (See Example 3, FIGS. 4A-C).

Whilst conceiving the present invention, the present inventorsuncovered. that processes inside the cell may be manipulated by changingthe extracellular pH of a solid buffer in contact therewith.Accordingly, the present inventors have shown that disruption ofcellular pH homeostasis may be effected by contacting cells with a solidbuffer comprising a pH which is different from the pH of theintracellular components. The contact results in the titration of theintracellular pH in the cytoplasm and generally leads to an alterationin a cellular process. Cell death may be effected when the pH of thebuffering material is beyond the viability range of pH for a specificcell.

U.S. Pat. Appl. Nos. 20050271780, 20050249695 and 20050003163 teachbactericidal polymers. The polymers as taught therein rely on the directcontact of the polymer with the cellular membrane since the bactericidalactivity originates from inclusion of cationic molecules, eitherimmobilized on surfaces of, or incorporated in polymeric structures. Thelevel of toxicity is strongly dependent on the surface concentration ofthe bactericidal entities. This requirement presents a strong limitationsince the exposed cationic materials can be saturated very fast in ionexchange reactions.

The solid buffers taught within are not restricted to cationic polymers,but anionic buffers as well, since the novel mechanism of the presentinvention does not rely on the penetration of cationic groups to disruptthe cell membrane, but relies on an overall bulk buffering effect. Thesolid buffers taught herein are not restricted by the surfaceconcentration of a bactericidal entity, since the cytotoxic activitythereof originates from their bulk properties and not just surfaceproperties.

Whilst reducing the present invention to practice, the present inventorsshowed that solid buffers may exert a cytotoxic effect on all celltypes, such as for example yeast cells (Example 4, Table 2), mammalianJurkat cells (Example 5, Table 3) bacterial cells (Example 11, Table 5)and fungal cells (Example 12).

The present inventors further demonstrated that the rate of cellmortality may be controlled by the choice of the pH value of the solidbuffer in contact with the cells, such that the rate of cell death canbe fine tuned by suitably modifying the pH values of the solid buffercontacting the cells (See e.g., Example 4, Table 2).

In addition, the present inventors showed that pH-induced cytotoxicityrequires direct contact of the cell with the solid buffer. Accordingly,physical barriers of a particular pore size may be attached to the solidbuffer, such that pH homeostasis is disrupted (altered) for cells of aparticular size only. In this fashion, cells of particular dimensionsmay be targeted leaving other cells unaltered (See Example 8).

Furthermore, the present inventors showed that a water permeable layerbeing disposed on an external surface of the buffering layer stillallows the solid buffer to exert its cellular affects since the waterpermeable layer allows the redistribution of ions and therefore does notdecrease the overall bulk effect of the solid buffer. Thus asillustrated in Example 14, solid buffers may be overlayed with open porepolymers and still exert cytotoxic effects.

Thus, according to one aspect of the present invention there is provideda method of generating a change in a cellular process of a target cellof a multicellular organism, the method comprising contacting the targetcell with a solid buffer, so as to alter an intracellular pH value in atleast a portion of said cell, thereby generating the change in acellular process of a target cell of a multicellular organism.

The cells of the present invention may be in any cellular environmente.g. isolated cells, a cell suspension, a cell culture, in a tissue, orin an organism. The cells may be healthy or diseased (e.g. tumor cells)or a combination thereof.

As used herein, the phrase “change in a cellular process” refers toeither an up-regulation of down-regulation in a cellular process.Exemplary cellular processes which may be changed according to thisaspect of the present invention, include but are not limited to rate ofcell death (apoptosis or necrotic cell death), cell differentiation,cell signaling cell growth, cell division, cell differentiation, cellproliferation, tumor growth, tumor vascularization, tumor metastases,tumor metastases migration and/or mobility, cellular mobility, organellefunction (including but not limited to, pseudopod formation, flagellarmotility, and the like) and molecular transport across various cellularand intracellular membranes and compartments.

According to a particularly preferred embodiment of this aspect of thepresent invention, the change in a cellular process results in cellkilling. Calibrating the solid buffer so that it is able to affect acytotoxic action is described hereinbelow.

As used herein, the phrase “multicellular organism” refers to anyorganism containing more than one cell. Exemplary multicellularorganisms include eukaryotes (e.g. mammals), and higher plants.

It will be appreciated that the solid buffers of the present inventionmay also be used to affect cellular processes in prokaryotic cells aswell—for example, fungi and gram positive and gram negative bacteria.

The term “Gram-positive bacteria” as used herein refers to bacteriacharacterized by having as part of their cell wall structurepeptidoglycan as well as polysaccharides and/or teichoic acids and arecharacterized by their blue-violet color reaction in the Gram-stainingprocedure. Representative Gram-positive bacteria include: Actinomycesspp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum,Clostridium perfringens, Clostridium spp., Clostridium tetani,Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcusfaecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae,Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum,Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex,Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacteriumhaemophilium, Mycobacterium kansasii, Mycobacterium leprae,Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacteriumsmegmatis, Mycobacterium terrae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Nocardia spp., Peptococcus niger,Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus,Staphylococcus auricularis, Staphylococcus capitis, Staphylococcuscohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcussaccharolyticus, Staphylococcus saprophyticus, Staphylococcusschleiferi, Staphylococcus similans, Staphylococcus wameri,Staphylococcus xylosus, Streptococcus agalactiae (group Bstreptococcus), Streptococcus anginosus, Streptococcus bovis,Streptococcus canis, Streptococcus equi, Streptococcus milleri,Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae,Streptococcus pyogenes (group A streptococcus), Streptococcussalivarius, Streptococcus sanguis.

The term “Gram-negative bacteria” as used herein refer to bacteriacharacterized by the presence of a double membrane surrounding eachbacterial cell. Representative Gram-negative bacteria includeAcinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans,Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroidesfragilis, Bartonella bacilliformis, Bordetella spp., Borreliaburgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp.,Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis,Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens,Enterobacter aerogenes, Escherichia coli, Flavobacteriummeningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilusspp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospiraspp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida,Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providenciarettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsiaprowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp.,Salmonella typhi, Serratia marcescens, Shigella spp., Treponemacarateum, Treponema pallidum, Treponema pallidum endemicum, Treponemapertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersiniaenterocolitica, Yersinia pestis.

As used herein, the phrase “solid buffer” refers to any solid materialwhich comprises a buffering capacity. A buffer capacity is defined asthe capacity of the buffer to resist changes in its pH when acids orbases are added to the buffer (titration) and is determined by theconcentration of H⁺ ions added per unit volume that may affect a changeof 1 pH unit in the buffer system. The buffer capacity of a system istypically derived from the coexistence in the system of dissociated andnon dissociated compounds capable of maintaining a constant supply of H⁺ions. Accordingly, any acidic or basic substance (i.e. ion exchangematerial) incorporated in an ion conductive or water/ion permeablematrix may be classified as a solid buffer. The buffer capacity of solidsubstances is typically derived from the presence of a plurality offunctional groups that can release or bind H⁺ and is determined by thedegree of saturation of these substances, namely, the H⁺ concentrationat which all of these functional groups interact.

Exemplary cationic ion exchange materials include, but are not limitedto sulfonic acid and derivatives thereof, phosphonic acid andderivatives thereof, carboxylic acid and derivatives thereof, phosphinicacid and derivatives thereof, phenols and derivatives thereof, arsenicacid and derivatives thereof and selenic acid and derivatives thereof.

Exemplary anionic ion exchange materials include, but are not limited tocompounds comprising teriary, secondary, primary and quaternary amines.

Examples of water permeable matrices include, but are not limited toopen pore polymers, open pore ceramics or gels.

Exemplary open pore polymers include, but are not limited to PVOH,cellulose and polyurethane.

Alternatively, the solid buffer may comprise a matrix which isintrinsically ion conductive. Examples of intrinsically ion conductingsolid buffers include, but are not limited to ionomers and polycationicmaterials.

Some examples of ionomers that have been commercialized are Nafion®™perfluorinated sulfonic acid membranes available from E. I. du Pont deNemours & Co., Inc. (Wilmington, Del.) and Surlyn®™ thermoplastic resin,also from DuPont.

Typically, the solid buffer is a polymer. It will be appreciated thatthere is a very wide variety of polymers that may be used as solidbuffers according to this aspect of the present invention. Below is alimited list of such polymers: Poly(4-vinyl-N-alkylpyridinium bromide),poly(methacryloyloxydodecylpyr-idinium bromide, N-alkylated poly(4-vinylpyridine), Poly(vinyl-N-hexylpyridinium), poly(N-alkyl vinylpyridine),poly(N-alkyl ethylene imine), poly(4-vinyl-N-alkylpyridinium bromide),poly(4-vinyl-N-hexylpyridinium bromide),Poly(1-(chloromethyl).sub.4-vinylbenzene,Poly(Dimethyloctyl[4-vinylphenyl]methylammonium chloride),Poly(Dimethyldodecyl[4-vinylphenyl]methylammonium chloride),Poly(Dimethyltetradecyll[4-vinylphenyl]methylammonium chloride), 50:50Poly(1-Chloromethyl)-4-vinylbenzene):Poly(Dimethyldodecyl[4-vinyl-phenyl]methylammonium chloride), 50:50Poly(1-Chloromethyl)-4-vinylbenzene):Poly(Dimethyloctyll[4-vinylp-henyl]methylammonium chloride), 50:50Poly(Dimethyldodecyll[4-vinylphenyl]methylammonium chloride):Poly(Dimethyloctyll[4-vinylphenyl]methylammonium chloride),Poly(Tributyl-[4-vinylphenyl]methylphosphonium chloride) andPoly(Trioctyl-[4-vinylphenyl]methylphosphonium chloride).

It will be appreciated that the solid buffer of the present inventionmay also comprise gel matrices such as a polyacrylamide and agarose gelmatrices which have been suitably prepared with appropriate buffers(e.g. with Immobiline™ acrylamido buffers). Amounts of immobilines pKbuffers used that produce gels of a particular pH are set forth in Table1 of the Example section below. The solid buffers of the presentinvention may also be ion exchange beads, polymer coated ion exchangebeads or ion exchange beads incorporated in an ion permeable matrix.

The term “contacting” as used herein refers to the positioning of thecell with respect to the solid buffer and is confined by the necessityof ions from the solid buffer to be conducted to the cell and vicaversa.

Thus, according to one embodiment of this aspect of the presentinvention, the cell and the solid buffer are in direct physical contactwith one another. For example, the solid buffer may contact the exteriorof the cell or adhere to the exterior of the cell. Alternatively thesolid buffer may be internalized by the cell by known processes ofinternalization of extracellular substances, such as, but not limitedto, phagocytosis, endocytosis, receptor mediated endocytosis,clathrin-coated pit or vesicle associated internalization processes,transferrinfection, and the like.

According to another embodiment of this aspect of the present invention,the solid buffer is separated from the cell by a water permeable layer.Such a water permeable layer would allow the flow of ions from the solidbuffer to the cell and vica versa and therefore would not impede withthe buffering capacity of the solid buffer. An exemplary water permeablelayer is PVOH, Ethyl cellulose, cellulose acetate, polyacrylamide, anymicroporous matrix without or with a hydrophilic additive etc.

The contacting may be effected in vivo, ex vivo (i.e. in cells removedfrom the body) and/or in vitro (e.g. in cell lines).

As mentioned hereinabove, the solid buffers of the present invention maybe formulated for generating a change in a particular cellular process.Typically, three properties of the solid buffer may be manipulated so asto allow the solid buffer to affect a cellular process—pH, buffercapacity and ion conductivity.

The following is an example of how a solid buffer may be selected inorder to affect (i.e. increase) the process of cell death:

1. The pH of the solid buffer should be out of range of viability of thecell. This range is specific for each type of cell and bacteria.Typically, a pH of less than 4 or greater than 8 of the solid bufferwill affect the pH stability of the cell.

It will be appreciated that the solid buffer may be formulated so thatit comprises a pH gradient. The gradient may be useful in providing agradual change in the biological effect of the solid buffer on thecells. For example, using such gradients on solid buffers may result inpart of the solid buffer having cytostatic effects on cells while otherregions of the solid buffer having cytotoxic effects.

It will be appreciated by those skilled in the art that variations inthe form, strength, position and overall pattern of such gradients maybe effected by suitably controlling ion exchange materials incorporatedin the matrix of the solid buffer, all of which are contemplated to beincluded within the scope of the present invention. Gradiented buffersmay be synthesized using Immobiline™ as described in the Examplessection hereinbelow.

Furthermore, it will be appreciated that the solid buffer of the presentinvention may also comprise a combination of cationic ion exchangematerials and anionic ion exchange materials arranged in a patternsuitable for effective killing. Thus the solid buffer may for examplecomprise a mixture of anionic and cationic beads. The beads may be ofthe same size or different size depending on the positioning of thetarget cells.

2. Since the generally accepted values for buffer capacity of thecytosol and most other cellular components is between 20-100 mMH⁺/liter.pH, therefore to cause titration of the cytosol, the buffercapacity of the solid buffer material should be higher than this value.A typical buffering capacity of the solid buffer that may be used tokill most cell types is about 100 mM H⁺/liter.pH or higher.

3. A change in the ion conductivity (proton conductivity) of a solidbuffer will affect the speed with which the solid buffer is able to killa cell. Typically, the ion mobility in a water permeable solid bufferwill be determined by the diffusional movement of protons in water andwill be of the order of about 10⁻⁸ m²/sec for the diffusion constant,this corresponding to a drift velocity of 0.1 mm/sec. Such solid bufferswill induce cell death in a cell in contact in a matter of seconds.

Thus, an exemplary method for killing a cell is by contacting the cellwith a solid buffer comprising a buffer capacity of about 50 mMH⁺/liter.pH and a pH capable of titrating the cell, thereby inducingcell death, the pH being generally greater than pH 8, or less than pH4.5.

Methods of measuring pH and determining buffering capacity are wellknown in the art.

It will be appreciated the plasma buffering capacity of cells and pH iscell-type specific and therefore manipulation of these parameters mayallow targeting to a particular cell type. For example, it is generallyaccepted that tumor cells are more alkaline than normal cells and thusin order to exert an optimal cytotoxic activity in tumor cells, thesolid buffer may have less (or no) effect on other cell types. Inaddition, each cell type has a particular membrane permeability andtherefore may inherently be more (or less) susceptible to the solidbuffers of the present invention.

As a further example, it is known that the buffer capacity of bacteriais higher than in mammalian cells but the vulnerability of bacteria totitration by buffers is higher since the mass of the buffering medium inbacteria is about three orders of magnitude smaller than in mammaliancells. This makes possible to use low buffer capacity solid buffers tokill bacteria without killing mammalian cells

One method of altering the pH and buffering capacity of solid buffers isby changing the concentration of an ion exchange material in a watersoluble (ion permeable) matrix. Alternatively, the concentration of theion exchange material may remain constant and the ion exchange materialmay be altered. An optimal solid buffer may be selected for killing acell of interest by testing a plurality of solid buffers comprisingdiffering pHs and buffering capacities on a mixture of cells includingthe cell of interest. The cell of interest may then be analyzed todetermine the optimal solid buffer. Methods of analyzing the cell ofinterest may include microscopy, immunohistochemistry or otherbiological assaying techniques known in the art.

Since the present invention contemplates using solid buffers to treatmedical conditions (e.g. one associated with a pathological cellpopulation), the solid buffer is typically administered to the body,either in vivo or ex vivo, and it is therefore particularly importantthat the solid buffers are able to selectively target specific celltypes.

Thus, according to an embodiment of this aspect of the presentinvention, the solid buffer may be attached to an affinity moiety, suchas an antibody, a receptor ligand or a carbohydrate. Examples ofantibodies which may be used according to this aspect of the presentinvention include but are not limited to tumor antibodies, anti CD20antibodies and anti-IL 2R alpha antibodies. Exemplary receptors include,but are not limited to folate receptors and EGF receptors. An exemplarycarbohydrate which may be used according to this aspect of the presentinvention is lectin.

The affinity moiety may be covalently or non-covalently linked to oradsorbed onto to the solid buffer using any linking or binding methodand/or any suitable chemical linker known in the art. The exact type andchemical nature of such cross-linkers and cross linking methods ispreferably adapted to the type of affinity group used and the nature ofthe solid buffer. Methods for binding or adsorbing or linking suchaffinity labels and groups are also well known in the art.

In accordance with one preferred embodiment of the present invention,the target cells may be metastasized cancer cells expressingidentifiable surface markers. If the pH and buffer capacity of the solidbuffer are selected to kill such cells upon contact, the affinitymoieties may be one or more antibodies directed against specific markersexpressed by such malignant cells.

Another method of targeting specific cell types (e.g. targetingprokaryotic cells and not eukaryotic cells) contemplated by the presentinventors is based on selectively preventing the physical contactbetween the solid buffer and particular cell types. Thus, according toanother embodiment of this aspect of the present invention, the solidbuffer is at least partially covered by a selective barrier. Forexample, if the surface of the solid buffer is covered or protected witha mechanical barrier having a controlled pore size (such as but notlimited to a filter e.g. nylon filter, having a selected pore size, or amesh with a selected opening size, or the like), it is possible toexclude cells above a certain size from attaching to or forming contactwith the solid buffer, while still allowing cells having a smaller sizeto enter the pores or to pass the mechanical barrier and to make contactwith the solid buffer.

Targeting the solid buffers of the present invention can also beachieved by using “passive” targeting. This exploits the enhancedpermeability of and retention of particles in tumor tissue due to leakyvasculature and lack of lymphatic drainage. It is known in the art thatthe selectivity for tumor for particles of size 200-600 nanometer isbetween 10 to 100 fold relative to healthy tissue. This particular typeof passive targeting may make use of particles which are notfunctionalized by recognition groups or moieties.

The solid buffer of the present invention can be administered to anorganism per se, or in a pharmaceutical composition where it is mixedwith suitable carriers or excipients.

As used herein, a “pharmaceutical composition” refers to a preparationof one or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

As used herein, the term “active ingredient” refers to the solid bufferaccountable for the intended biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier,” which may be usedinterchangeably, refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered compound. Anadjuvant is included under these phrases.

Herein, the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found inthe latest edition of “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., which is herein fully incorporated byreference.

The solid buffer of the present invention may be formulated as particlesor beads and may be manufactured in mean sizes within the range ofseveral nanometers to few millimeters and larger.

The solid buffer may be attached on the particle surface or encapsulatedwithin the particles. It will be appreciated that if the solid buffer isheld within the particle, the encapsulating particle must be made of anion conducting material to allow the flow of ions between the solidbuffer and the cell. Exemplary particles include, but are not limited topolymeric particles, microcapsules liposomes, microspheres,microemulsions, nanoparticles, nanocapsules and nanospheres.

The solid buffers of the present invention may also be coated bybiodegradable coatings in order to improve selectivity and preventactivity while in circulation. Exemplary biodegradable coatings includePolyethylenimine (PEI) coatings, polyethylene glycol (PEG) coatingsmodified gelatin coating or any other suitable coating material.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal, or parenteraldelivery, including intramuscular, subcutaneous, and intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For topical administration, the solid buffer of the present inventionmay be formulated as a gel, a cream, a wash, a rinse or a spray. Thismay be applied when the solid buffer is administered topically to asubject or onto any solid surface.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries as desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethylcellulose, and sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents, such ascross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, or carbon dioxide. In the case of apressurized aerosol, the dosage may be determined by providing a valveto deliver a metered amount. Capsules and cartridges of, for example,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with, optionally, anadded preservative. The compositions may be suspensions, solutions, oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water-based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters such as ethyl oleate, triglycerides, orliposomes. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe active ingredients, to allow for the preparation of highlyconcentrated solutions.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, for example, conventional suppository bases such as cocoabutter or other glycerides.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a “therapeutically effective amount” means an amountof active ingredients (e.g., a nucleic acid construct) effective toprevent, alleviate, or ameliorate symptoms of a disorder (e.g.,ischemia) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, the dosage orthe therapeutically effective amount can be estimated initially from invitro and cell culture assays. For example, a dose can be formulated inanimal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration, and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl, E. et al. (1975), “The PharmacologicalBasis of Therapeutics,” Ch. 1, p. 1.)

Dosage amount and administration intervals may be adjusted individuallyto provide sufficient plasma or brain levels of the active ingredient toinduce or suppress the biological effect (i.e., minimally effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks, oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser device may also be accompaniedby a notice in a form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may includelabeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a preparation of the invention formulated in apharmaceutically acceptable carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition, as further detailed above.

It will be appreciated that the present invention also contemplatescoating a solid surface or material with the solid buffer of the presentinvention. The term “surface” as used herein refers to any surface ofany material, including glass, plastics, metals, polymers, and like. Itcan include surfaces constructed out of more than one material,including coated surfaces.

The solid buffer may be attached to a surface using any method known inthe art including spraying, wetting, immersing, dipping, painting,ultrasonic welding, welding, bonding or adhering or otherwise providinga surface with the solid buffer of the present invention. The solidbuffers of the present invention may be attached as monolayers ormultiple layers.

An exemplary solid surface that may be coated with the solid buffers ofthe present invention is an intracorporial or extra-corporial medicaldevice or implant.

An “implant” as used herein refers to any object intended for placementin a human body that is not a living tissue. The implant may betemporary or permanent. Implants include naturally derived objects thathave been processed so that their living tissues have been devitalized.As an example, bone grafts can be processed so that their living cellsare removed (acellularized), but so that their shape is retained toserve as a template for ingrowth of bone from a host. As anotherexample, naturally occurring coral can be processed to yieldhydroxyapatite preparations that can be applied to the body for certainorthopedic and dental therapies. An implant can also be an articlecomprising artificial components.

Thus, for example, the present invention therefore envisions coatingvascular stents with the solid buffers of the present invention. Thesolid buffers may repel or attract specific type of proteins in cellswhich may affect the cell cycle of endothelial cells in contact with thesurface to reduce or prevent restenosis, or general type of implantscoated by the method of the present invention to achieve beneficialeffect in the integration of the implant with tissue.

Another possible application of the solid buffers of the presentinvention is the coating of surfaces found in the medical and dentalenvironment.

Surfaces found in medical environments include the inner and outeraspects of various instruments and devices, whether disposable orintended for repeated uses. Examples include the entire spectrum ofarticles adapted for medical use, including scalpels, needles, scissorsand other devices used in invasive surgical, therapeutic or diagnosticprocedures; blood filters, implantable medical devices, includingartificial blood vessels, catheters and other devices for the removal ordelivery of fluids to patients, artificial hearts, artificial kidneys,orthopedic pins, plates and implants; catheters and other tubes(including urological and biliary tubes, endotracheal tubes,peripherably insertable central venous catheters, dialysis catheters,long term tunneled central venous catheters peripheral venous catheters,short term central venous catheters, arterial catheters, pulmonarycatheters, Swan-Ganz catheters, urinary catheters, peritonealcatheters), urinary devices (including long term urinary devices, tissuebonding urinary devices, artificial urinary sphincters, urinarydilators), shunts (including ventricular or arterio-venous shunts);prostheses (including breast implants, penile prostheses, vasculargrafting prostheses, aneurysm repair devices, heart valves, artificialjoints, artificial larynxes, otological implants), anastomotic devices,vascular catheter ports, clamps, embolic devices, wound drain tubes,hydrocephalus shunts, pacemakers and implantable defibrillators, and thelike. Other examples will be readily apparent to practitioners in thesearts.

Surfaces found in the medical environment include also the inner andouter aspects of pieces of medical equipment, medical gear worn orcarried by personnel in the health care setting. Such surfaces caninclude counter tops and fixtures in areas used for medical proceduresor for preparing medical apparatus, tubes and canisters used inrespiratory treatments, including the administration of oxygen, ofsolubilized drugs in nebulizers and of anesthetic agents. Also includedare those surfaces intended as biological barriers to infectiousorganisms in medical settings, such as gloves, aprons and faceshields.Commonly used materials for biological barriers may be latex-based ornon-latex based. Vinyl is commonly used as a material for non-latexsurgical gloves. Other such surfaces can include handles and cables formedical or dental equipment not intended to be sterile. Additionally,such surfaces can include those non-sterile external surfaces of tubesand other apparatus found in areas where blood or body fluids or otherhazardous biomaterials are commonly encountered.

Other surfaces related to health include the inner and outer aspects ofthose articles involved in water purification, water storage and waterdelivery, and those articles involved in food processing. Thus thepresent invention envisions coating a solid surface of a food orbeverage container to extend the shelf life of its contents.

Surfaces related to health can also include the inner and outer aspectsof those household articles involved in providing for nutrition,sanitation or disease prevention. Examples can include food processingequipment for home use, materials for infant care, tampons and toiletbowls.

As illustrated in Example 15, the solid buffers of the present inventionmay be used to enhance the antibacterial activity of a wound dressing.Similarly, the solid buffers of the present invention may be used toenhance the antibacterial activity in sutures, cloth, fabrics and woundointments.

In accordance with another embodiment of the present invention, thesolid surface may be a microscopic slide, a culturing hood, a Petri dishor any other suitable type of tissue culture vessel or container knownin the art.

As used herein, the term “about” refers to plus or minus 10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Distribution of Proteins in a pH Gradient

The following experiment was carried out in order to ascertain whetherproteins comprise specific pH characteristics.

Materials and Methods

Gel preparation: Four immobiline gel strips measuring approximatelyseven centimeters were used. Each strip was cut from an ampholinecontaining polyacrylamide based gel having a pH gradient from 4-9prepared as is known in the art (the gel contained 4% polycacrylamideand 5% bisacrylamide cross linker).

Protein solution preparation: Four different protein solutions wereprepared. The first solution contained 1.0 mg/ml myoglobin (commerciallyavailable from Sigma, USA—catalogue number M-0630) in DDW. The secondsolution contained 1.0 mg/ml myoglobin in DDW including a finalconcentration of 8M Urea for protein denaturation. The third solutioncontained 1.0 mg/ml phycocyanin (commercially available from Sigma, USAas Catalogue Number P-2172) in DDW. The fourth solution contained 1.0mg/ml phycocyanin in DDW including a final concentration of 8M Urea forprotein denaturation. All protein solutions had a pH of about 7.0.

Experimental Procedure: Ten ml of each of the above described proteinsolutions were placed in a Petri dish and a strip of gel was immersedinto each one. The Petri dishes were covered and the gels were incubatedfor 3-5 days at room temperature. At the end of the incubation period,the gel strips were removed from the Petri dishes, carefully blottedfrom excess liquid and scanned in an Epson flatbed office scanner.

Results

As illustrated in FIGS. 1A-B and 2A-B, the proteins were adsorbeddifferently into different regions of the gel strips according to the pHat the different regions of the strip.

Myoglobin has a pI≈6 and Phycocyanin has a pI≈4.2. While the shift ofthe spatial (and pH dependent) distribution curve for myoglobin betweennative and denatured protein is rather small (FIGS. 2A-2B), a verystrong shift (difference in spatial distribution of absorbance as afunction of pH along the gel strip) is observed for the much largerprotein Phycocyanin (FIGS. 1A-1B).

Conclusion

The distribution of a protein in a pH gradient presents a property whichis specific for each tested protein and may be presented as a pHcharacteristic of the protein.

Example 2 Redistribution of Proteins Across a Gel Membrane According totheir pH Characteristics

Materials and Methods

Gel preparation: A rectangular chamber was divided into threecompartments by placing two gel membranes, formed as 2 mm thickpolyacrylamide-based gel slabs, as illustrated in FIGS. 3A-B. The gelswere prepared by addition of 10 μl of Imobiline™ (Amersham), 0.5 μlammonium persulfate (APS), 0.25 μl TEMED (1:10), 10% polycarylamide and5% bisacrylamide. One gel membrane was prepared for pH 4 (acidic,Polyacrylamide with Immobilines) and the other gel membrane was preparedfor pH 6 (basic, Polyacrylamide with Immobilines).

Protein solution preparation: Myoglobin and Phycocyanin were dissolvedin doubly deionized water (DDW) each at a concentration of 0.1gram/Liter (g/L).

Experimental Procedure: 300 μl of each protein solution was placed intothe central chamber bordered by the two membranes. The chamber on theacidic side was filled by a buffer solution of 1 mM of glutamic acid(pH=3.8) and the chamber at the basic side was filled with 1 mM solutionof TRIS (pH=8.3). The chamber was left undisturbed at room temperature.After several days, the chamber was visually observed and alsophotographed (top view) using a digital camera.

Results

At the beginning of the experiment the solution in the middlecompartment 2 of the multi compartment chamber described above has adark color resulting from the combined absorbance of the myoglobin andphycocyanin present in the middle compartment 2, while there was hardlyany color observed in the compartments labeled 1 and 3 which containedthe acidic (1 mM of glutamic acid; pH=3.8) buffer and the basic buffer(1 mM solution of TRIS; pH=8.3), respectively, as illustrated in FIG.3A.

As illustrated in FIG. 3B, by the end of the experiment, the solution inthe middle compartment 2 of the multi compartment chamber describedabove had a much fainter magenta-like color resulting from the combinedabsorbance of a much lower concentration of myoglobin and phycocyaninleft therein. A strong reddish color was observed in the compartmentlabeled 1 which contained the acidic (1 mM of glutamic acid; pH=3.8)buffer into which a large portion of the myoglobin migrated. A strongbluish color was observed in the compartment labeled 3 which containedthe basic buffer (1 mM solution of TRIS; pH=8.3), into which a largeportion of the phycocyanin migrated.

Conclusion

An almost complete redistribution and separation of the two coloredproteins occurred governed by the different pH values in thecompartments separated by the Imobiline™ membranes.

Example 3

The following experiment was carried out to demonstrate the feasibilityof affecting intracellular distribution of a cytoplasmic protein withina living functioning cell.

Materials and Methods

HeLa cells were transfected to express GFP in their cytosol. Followinglysis, the extracted proteins were tested to determine the pH region ofmaximum accumulation of the GFP protein as described in Example 1 above.The region of maximum accumulation of the GFP protein (as determined bylocating the peak fluorescence on the scanned gel strip) was found to beat about pH=9.

Commercial polyacrylamide beads having a mean diameter of approximately50 microns (Biogel P10, Cat. No. 1504140, Biorad, USA) were soaked in asolution of a copolymer of polyacrylamide and immobilines at pH=9(prepared as detailed in Example 2 hereinabove). The Imobiline™polyacrylamide solution was allowed to chemically polymerize followingwhich an aqueous suspension of the resulting beads was added to a cellculture of the HeLa cells expressing GFP. Some of the cells attachedthemselves to the beads. The mixture of cells and beads was thenimmobilized by casting an agarose solution (Low melt agarose, catalogueNumber 1620019 Biorad, USA and having a melting point of about 36° C.)on the cells and the beads, and allowing the agarose to cool to about25° C. A cell in contact with a bead was observed under a fluorescencemicroscope (Axioscope 2 Fluorescence Microscope, Zeiss, Germany) and thechange of distribution of the GFP was visually and photographicallymonitored over a period of 30 minutes.

Results

As may be seen in FIGS. 4A-C, the fluorescence intensity at the point ofattachment of the cell to the bead was about fifty times higher 30minutes following initial attachment than the initial intensity in thecell as measured at time zero. This measured intensity accounts for amajor fraction of the GFP in the cell. A similar phenomenon was observedin several cells which were attached to the beads.

Control experiments with similar beads having coating with pH=7 (notshown) did not show any change in the distribution pattern of GFP incells attached to the beads and similarly observed.

Conclusion

The above experimental observations clearly demonstrate that a localizedprotein (GFP) accumulation or redistribution mechanism based on pHpartitioning may be induced in a living cell and that it is possible togenerate a concentration gradient or a localized concentration of anintracellular protein using contact with a material or object which hasa controlled pH at it's surface. The experiment also demonstrates thatthis property can be utilized to cause redistribution of one or moreproteins in a living cell.

Example 4 Effect of pH on Cytotoxicity of Yeast Cells

This experiment was performed to test the cytoxicity of pH modifiedsurfaces on yeast cells.

Materials and Methods

The bottom of nine plastic Petri dishes were coated with a 0.5 mm thickpolyacrylamide gel with immobilines (acrylamido buffers), each gelhaving a different pH from the preceding gel by about 1 pH unit. Thecoating of the first dish was a pH 3 acrylamido Imobiline™ buffer gel,the coating of the second dish was a pH 4 acrylamido Imobiline™ buffergel, the coating of the third dish was a pH 5 acrylamido Imobiline™buffer gel etc, . . . , and the coating of the ninth dish was a pH 11acrylamido Imobiline™ buffer gel.

The coating was prepared by standard polymerization methods as is knownin art. The composition of imobilines is set forth in Table 1hereinbelow:

TABLE 1 IMMOBILINE pK BUFFERS USED (μl) pH 3.6 4.6 6.2 7.0 8.5 9.3 3.00256 0 4 0 0 0 4.00 276 103 59 0 0 170 5.00 295 200 111 0 0 331 6.00 295200 111 0 0 331 7.00 130 532 90 188 0 551 8.00 0 605 0 273 147 476 9.00219 0 212 231 72 284 10.0 0 40 0 1138 85 237 11.0 0 1 0 1345 99 335 Thenumbers in table 1 are given as μl of starting material (having aconcentration of 100 mM) used to prepare 10 ml of pH solution byaddition of DDW.

1-2 Million yeast cells Sacharomices (commercially available baker'syeast), suspended in tissue culture medium (Roswell Park Memorial TissueCulture Media, RPMI-1640 Dutch Mod. 01-1-7-1) were placed in each of thePetri dishes. The cells sedimented to the bottom of the dish and came incontact with the polyacrylamide surface and were left in the dish for apreset time as indicated Table 2 hereinbelow. Following the indicatedcontact time, the cells were stained using Trypan Blue and the number ofdead cells was estimated for each dish.

Results

Table 2 below lists the cell mortality data (as % of total cells) at theindicated pH and exposure time.

TABLE 2 Time (hours) pH 0.25 0.5 1 2 4 6 12 3 50 100 100 100 100 100 1004 25 25 50 55 80 100 100 5 2.5 15 25 45 65 95 97.5 6 1.5 15 25 35 50 5560 7 1.5 2.5 5 3 1.5 5 1 8 2.5 2.5 5 3.5 1.5 4 2.5 9 5 55 45 55 70 80 8510 25 50 50 60 85 95 99 11 50 100 100 100 100 100 100 Control 1.5 2.53.5 2.5 1.5 4 3.5 (Pure PA)

As may be seen from Table 2, at extreme pH values (pH 3, pH 4, pH10, andpH 11) the cells die within a relatively short time of contact with thepH controlling substrate. At pH 7 and pH 8, no significant cell toxicityis observed even after a prolonged time. At intermediate pH values (inthe range of pH 5-9), a time dependent toxicity is observed.

Conclusion

The only parameter that was changed in the gels was the composition ofthe acrylamido buffers. Since the gel is very stable under aqueoussoaking, no release of any kind of toxic agent into the cell culturemedia can be envisioned. Therefore, the cell toxicty as observed is mostprobably due to the redistribution of ions (charged proteins, hydrogenions, potassium ions, and other intracellular ions) in the cell oncontact with the surface of the pH controlling acrylamide gels used inthe experiment. This assumption is further supported by the fact that ifone compares the compositions and toxicity as observed at pH 3, 4, 5 and6 the concentration of the highly acidic component is almost constantwhile the toxicity changes are very significant. The same can beobserved on the basic side where the concentration of the most basiccomponent changes only slightly for pH 11, 10, 9 and 8 whereas thetoxicity changes significantly.

This observation proves that the toxicity is not the result ofincorporation of the highly anionic or cationic species as claimed inprior art but the result of the bulk pH property.

The results of this experiment further demonstrate that the rate of cellmortality (delayed cytotoxicity effect) can be controlled by the choiceof the pH value in the pH controlling substance or substrate in contactwith the cells, and that such effects (the rate of cell death) can befine tuned by suitably modifying the pH values of the surface orsubstrate contacting the cells.

Example 5 Effect of pH Induced Cytotoxicity in Jurkat Cells

Materials and Methods

Jurkat cells, Clone E6-1 were grown in RPMI 1640 supplemented with 2 mML-glutamine, 10 mM HEPES, 10 mM sodium pyruvate and 10% PBS. The cellswere exposed to varying pH surfaces as described for yeast cellshereinabove (Example 4).

Results

Table 3 below lists the cell mortality data (as % of total cells) at theindicated pH and exposure time. The results demonstrate high celltoxicity of the surfaces having low and high pH.

TABLE 3 TIME (hours) pH 1 hour 2 hours 3 hours 3 15 90 90 4 8 20 80 5 115 3 6 0 0 0 7 0 3 3 8 5 10 7 9 9 9 6 10 — — — 11 3 12 80 Control, PurePA 0 0 0

Example 6 Absorption Characteristics of Yellow Fluorescent Protein (YFP)

Materials and Methods

1 μg of H1299 lung cancer cells expressing a yellow fluorescent protein(Source—Phialadium sp. SL-2003) were lysed. The extracted proteins weretested to determine the pH region of maximum accumulation of the YFP onan IPG strip (Amersham Biosciences, Immobiline™ Dry Strip pH 3-10). Thestrip was immersed in the solution for 22 hours, following which it wasscanned with a UV scanner of a Zeiss Axiscope 2 Plus, UV microscope.

Results

As can be seen in FIG. 5, a pH range of 9.5-10 showed the strongestaccumulation of the YFP.

Example 7 Physical or Mechanical Barriers Prevent pH InducedCytotoxicity

The following experiment was designed in order to ascertain whetherpH-induced cytotoxicity requires direct contact of the cell with thesurface of the pH controlling substrate.

Materials and Methods

A 0.5 mm thick layer of pH 3 immobiline Polyacrylamide gel (IPG) wascast on the bottom of a Petri dish. A 10 μm thick nylon filter with a 2μm mean pore size (commercially available from Nalgene, USA) was placedin close contact with the surface of the IPG layer.

A suspension of 0.2 million yeast cells in tissue culture medium(Roswell Park Memorial Tissue Culture Media, RPMI-1640 Dutch Mod.01-1-7-1), was placed in the Petri dish and the cells were left tosediment for six hours. At the end of the six hour sedimentation period,the cells were stained with Tryptan Blue.

Results

The number of dead cells counted was approximately 5% of the totalnumber of cells counted.

Conclusion

The nylon filter interposed between the cells and the surface of the pHcontrolling substrate, prevented the pH-induced cytotoxicity.

Example 8 Differential Cell Toxicity Device

In order to further establish that direct contact between a cell and thepH controlling substrate is required for pH-induced cytotoxicity, afilter allowing bacteria cells to be in contact with the substrate,while not allowing yeast cells to be in contact with the substrate wasused as follows:

Materials and Methods

A 0.5 mm thick layer of pH 3 immobiline Polyacrylamide gel (IPG) wascast on the bottom of a Petri dish. A 10 μm thick nylon filter with a 2μm mean pore size, as described in Example 6 above, was placed in closecontact with the surface of the IPG layer. A mixture of E.Coli (100units/microliter) and Yeast cells (1 million/ml) suspended in 0.5 mls ofcell culture medium was placed in the Petri dish on top of the nylonfilter and the dish was incubated for a period of 12 hours at 37° C.Following the incubation period, the culture medium was sampled forbacterial colonies on McConkey Agar. The yeast cells were then stainedwith Tryptan Blue for performing dead cell count.

Results

No bacterial colonies were detected and no significant yeast cellmortality was observed.

Conclusion

The results of this experiment demonstrate that bacterial cells whichwere in contact with the cytotoxic agent were killed, whereas the yeastcells which were not in contact with the cytotoxic agent remained alive.The results of this experiment further demonstrate the bacterio-toxicproperty of the pH controlling substrate.

Example 9 pH Induced Cytotoxicity in Jurkat Cells

In order to establish whether pH-induced cytotoxicity occurs in Jurkatcells, the following experiment was performed.

Materials and Methods

A suspension of Polyacrylamide based beads having an approximate meanbead size of about one micron was prepared from apolyacrylamide+Immobiline™ mixture having a pH of 9.0. The beads wereadded to one million Jurkat cells suspended in 1 ml of tissue culturemedium such that the ratio of beads to cells was approximately 20 beadsper Jurkat cell. Aliquots were drawn out at 0.5, 1.0 and 2.0 hoursfollowing addition of the beads to the cell suspension. The cells werestained with Tryptan Blue dye and the number of dead cells and totalcells was counted.

Results

At 0.5 hours following bead addition, the fraction of dead cells in thesample was 5%. At 1.0 hour following addition of the beads to the cells,the fraction of dead cells in the sample was 10%. At 2.0 hours followingaddition of the beads to the cells, the fraction of dead cells in thesample was 27%.

Example 10 Cytotoxic Effect of Nafion

Sulfonated tetrafluorethylene copolymers (e.g. Nafion) are acidic(anionic charged) bioactive polymers with strong buffering propertiesand high buffering capacities. These types of films consist of asubstrate (e.g. polymethylacrylate, nylon or polyester) and a sulfonatedpolymer as an active layer. Nafion is not recognized as cytotoxic orbactericidal and is generally used as an ion conductive electrode infuel cell applications. The following toxicity tests were performed toascertain whether Nafion is toxic to cells.

Materials and Methods

1 million Jurkat cells in PBS buffer were deposited on a 1 cm square ofa nafion commercial membrane (NAFION 117, Perfluorinated membrane,Sigma, 274674-1EA). In order to differentiate between live and deadcells, the membrane was stained with 1 μl of 1 μg/μl of Propidium iodideor trypan Blue.

Results

Following a 10 minute exposure to the nafion, more than 95% of cellswere dead as seen in FIGS. 6A-B.

Example 11 Bacteriotoxicity and Cytotoxicity of Laminates

Materials and Methods

Laminate samples: laminate samples consisted of films coated on a 110 μmpolyester base.

Series BIOACT 13, 15 and 16:

BIOACT 16: 110 μm polyester base+a primer layer of acrylic modifiedpolyurethane.

BIOACT 13: 110 μm polyester base+a primer layer of acrylic modifiedpolyurethane+“active” cationic submicron silica in PVOH binder (w/wratio 4:1); total coating weight of 0.97 g/m², coating pH 4.06.

BIOACT 15: 110 μm polyester base+a primer layer of acrylic modifiedpolyurethane+“active” cationic polyurethane polymer in PVOH binder (w/wratio 4:1); total coating weight of 0.76 g/m², coating pH 4.

Uncoated sample was provided as control.

Series MVC/HT/56 A, B and C: This set of laminates was based on theincorporation of p-Toluenesulphonic acid salt (pH 3) ofpoly(diethylaminoethylmethacrylate) as the active component of thecoatings.

MVC/HT/56 A: 110 μm polyester base+PVOH+p-Toluenesulphonic acid salt.The total dry coating weight 0.9 gsm (˜0.9 microns) of which the drycoat weight of the active component is 0.6 gsm.

MVC/HT/56 B: Identical to MVC/HT/56 A, but a different batch.

MVC/HT/56 C: 110 μm polyester base+PVOH+p-Toluenesulphonic acid salt.The total dry coating weight 0.58 gsm (˜0.5 microns) of which the drycoat weight of the active component is 0.24 gsm.

MVC/HT/56 D Identical to MVC/HT/56 A, but a different batch.

Preparation of live and dead Bacterial Suspensions: 10 ml of E. coli DH5were grown to late log phase in LB broth. 1 ml of the culture wasconcentrated by centrifugation at 5000 rpm for 5 minutes. The pellet wasresuspended in 100 μl of 0.85% NaCl. 50 μL of this suspension was addedto 950 μl of 0.85% NaCl (for live bacteria) or 850 μl of 70% 2-propanol(for dead bacteria). Both samples were incubated at RT for 1 hour,following which they were pelleted by centrifugation at 5000 rpm for 5minutes. The obtained pellets were resuspended in 500 μl of 0.85% NaCland re-centrifuged. Finally, both pellets were resuspended in 50 μl0.85% NaCl.

Staining of Live and Dead bacterial suspensions: Staining was performedwith LIVE/DEAD® BacLight™ Bacterial Viability Kit (Molecular probes).With a mixture of SYTO9 and propidium iodide stains, bacteria withintact cell membranes stain fluorescent green, whereas bacteria withdamaged membranes stain fluorescent red. Essentially, 2 μl of SYTO 9dye, 1.67 mM/Propidium iodide and 1.67 mM Component A Was mixed with 2μl of 1.67 mM/Propidium iodide, 18.3 mM Component B. 0.15 μl of the dyemixture was added to 50 μl of the bacterial suspensions. 2.5 μl of thestained bacteria was trapped between a slide and coverslip. Live andkilled cells were observed under a fluorescence microscope.

Antibacterial activity testing of films: Two series of tests wereperformed using non activated (from the shelf) films and films treatedfor 20 minutes in a 1M NaCl solution. In both of them the antibacterialactivity was estimated by counting under a fluorescent microscope thenumbers of dead and live stained bacteria in the sample deposited on thebioactive film.

Cytotoxic activity testing of films: Live and dead Jurkat cells werecounted following exposure to the bioactive films by the followingprocedure: 0.15 μl of the dye mixture was added to 1 million Jurkatcells in 50 μl PBS. 2.5 μl of the stained cells were trapped between anactivated film and coverslip. Live and dead cells were observed under afluorescence microscope.

Results

Antibacterial activity testing of MVC HT 56A, B, C and D: Following a 30minute incubation of Jurkat cells with the laminates describedhereinabove, live (moving) cells were observed in control, MVC/HT/56/Bfilm and MVC/HT/56/D film, which under a green filter (5-2) were greenor reddish. In comparison, after 1 minute of incubation on MVC/HT/56/Aand/56C laminates all cells were attached and under a green filter wereobserved as red.

Cytotoxic activity testing of MVC HT 56A, B, C and D: As can be seenfrom FIG. 7 and Table 4 herein below interaction of Jurkat cells with56/A and 56/C films differs from 56/B and 56/D.

TABLE 4 Time, min 1 10 20 30 45 60 % of dead Jurkat cells wo carrier 2.38.9 12.6 14.4 16.3 20.2 control 6.3 7.1 11 12 15 15.7 56A 3.2 12 25.443.1 32.8 40 56B 3.5 17 14.4 56C 18.5 46.1 64.6 56D 13 25.3 27

Antibacterial activity testing of BIOACT 13, 15 and 16: Following 45minutes of incubation on BIOACT 13, 50-70% of E.coli cells were dead. Atthe same time, 20-40% of E.coli were dead following incubation on BIOACT15. E.coli cells were not attached to BIOACT 16. Following 20-30 minutesof incubation on BIOACT 15, almost all E.coli cells were dead.Evaluation of the attachment of these cells was difficult due to a highbackground noise level.

Cytotoxic activity testing of BIOACT 13, 15 and 16: The results from 3separate experiments are illustrated below in Table 5 and are presentedas the number of green:red Jurkat cells and percentage of green overall.

TABLE 5 Ex. 1 Ex. 2 Ex. 3 Ex. 1 Ex. 2 Ex. 3 (1 minute) (1 minute) (1minute) (10 minutes) (10 minutes) (10 minutes) wo 81/13 (13.8) 288/8(2.7) 308/9 (2.8) 120/20 (14.3) 300/29 (8.8) 260/21 (7.5) carrier 13250/15 (5.7) 400/26 (6.1) 360/26 (6.7) 28/14 (33.3) 80% 60% 15 160/14(8.1) 280/12 (4.1) 280/8 (2.8) 200/160 (44.4) 81/5 (6.2) 160/40 (20) 16120/13 (9.8) 320/7 (2.1) 376/29 (7.2) 144/25 (14.8) 100/6 (5.7) 364/30(7.6) 110 100/5 (4.8) 250/6 (2.3) 320/13 (3.9) 23/58 (71.6) 216/15 (6.5)66/28 (29.5)

Table 6 hereinbelow summarizes the results from the three experiments asthe percentage of red cells. FIGS. 8A-D illustrate a typical experimentfollowing exposure of Jurkat cells to Bioact 13

TABLE 6 1 minute 10 minute wo carrier 6.4 10.2 13 6.2 57.8 15 5 23.5 166.4 9.4 110 3.7 35.9

Conclusion

Interaction of E.coli cells and Jurkat cells with 56/A and 56/C filmsdiffers from 56/B and 56/D. In the BIOACT 13, 15 and 16 series, BIOACT13 showed the highest cytotoxic and anti-bacterial activity.

Example 12 Bactericidal Activity of Nafion and Polyacrylamide pH Gels

The following toxicity tests were performed to ascertain whether Nafionand other films are toxic to bacteria.

Materials and Methods

Six types of plastic films were tested for bactericidal effects:

-   1. Nafion (commercial, Dupont);-   2. Nafion (commercial, Dupont);-   3. 500 micron thick polyacrylamide with immobilines on polyester    base pH 10;-   4. Same as 3 at pH 9;-   5. Polyuretane film (commercial);-   6. 500 micron poyacrylamide on polyester pH 5; Control-polyester    Film

Testing Staph. Aureus, Staph. Spp, Strept. Beta-hemolit.gr.A and Strept.Beta-hemolit.gr.G: The viability of these bacteria was tested on bloodagar using the “sow” method. Essentially, 0.01 ml of microbial liquidculture was spread on blood agar using a special bacterial loop. Each ofthe six plastic films (10 mm×10 mm) was placed on the testing plate withactive side down. Following overnight incubation at 37° C., the numberof colonies was evaluated. The test and control groups were compared.

Testing total microbial and fungal agents: The total anti-microbial andanti-fungal effect of the above films was tested on Saburo agar usingthe “sedimentation” method. Uncovered plates with Saburo agar wereplaced for 8 hours in the open. Each of the six plastic films (10 mm×10mm) was placed on the testing plate with active side down. Followingovernight incubation at 37° C., the number of colonies was evaluated.The test and control groups were compared.

Results

The effects of the sheets of the present invention on Staphaureus growthare summarized in Table 7. The effects of the sheets of the presentinvention on Staph. Spp growth are summarized in Table 8. The effects ofthe sheets of the present invention on Strept. Beta-hemolit.gr.A growthare summarized in Table 9. The effects of the sheets of the presentinvention on Strept. Beta-hemolit.gr.G growth are summarized in Table10. The effects of the sheets of the present invention on totalmicrobial and fungi agents are summarized in Table 11.

TABLE 7 Staph. aureus growth Control sheet (agents/ml) (agents/ml) 1  2*10³ >10⁶ 2 <10³ >10⁶ 3 <10³ >10⁶ 4 4.5*10⁴ >10⁶ 5 9.9*10⁴ >10⁶ 67.36*10⁵  >10⁶

TABLE 8 Staph. Spp Control sheet (agents/ml) (agents/ml) 1 <10³ >10⁶ 2<10³ >10⁶ 3   9*10³ >10⁶ 4  2.7*10⁴ >10⁶ 5 4.29*10⁵ >10⁶ 6 8.03*10⁵ >10⁶

TABLE 9 Strept betta-hemolit. gr. A Control sheet (agents/ml)(agents/ml) 1   3*10³ >10⁶ 2 <10³ >10⁶ 3 <10³ >10⁶ 4  1.1*10⁴ >10⁶ 56.71*10⁵ >10⁶ 6 8.59*10⁵ >10⁶

TABLE 10 Strept betta-hemolit. gr. G Control sheet (agents/ml)(agents/ml) 1 2*10³ >10⁶ 2 5*10³ >10⁶ 3 4*10³ >10⁶ 4 2.1*10⁴   >10⁶ 58.61*10⁵   >10⁶ 6 7.8*10⁵   >10⁶

TABLE 11 Total microbial and fungi Control sheet agents (colonies)(colonies) 1 3 22 2 2 18 3 1 17 4 9 19 5 19 21 6 19 19

Conclusion

Both Nafion and sheet no. 3 (the 500 micron thick polyacrylamide withimmobilines on polyester base pH 10) showed high antibacterialactivities and total antimicrobial and antifungal activities.

Example 13 Shelf Life Tests on Milk

The films of the present invention were tested for their effect on milkshelf life.

Materials and Methods

Pasteurized, homogenized milk was used in order to test milk stabilitywith the films of the present invention. In both sets of experiments themilk was UV treated.

Test 1: Seven empty 35 mm Petri plates were filled to the top with freshmilk. Six plates were covered with the films of the present invention,so that their active side contacted the milk w/o air between them. Theseventh plate was used as a control. Plates were placed on the table atroom temperature for six days. Each day the pH of the plate was tested.In order to compensate for evaporation, sterile DDW was added each day.The total volume of added DDW was less then 5% of the total milk volumeand therefore was not expected to influence pH dynamics. This experimentwas repeated twice.

Test 2-14 day test with Nafion: This test was performed with commercialNafion as the active material (layer). Pasteurized, homogenized milk(w/o antibiotics) was used in order to test milk stability. Three empty35 mm Petri plates were filled with fresh milk up to the top. Two werecovered with Nafion, so that active side contacted the milk w/o airbetween them. The third plate was used as control. Plates were placed onthe table at room temperature for fourteen day. Each day pH of the platewas tested. In order to compensate for evaporation, sterile DDW wasadded each day. The total volume of added DDW was less then 5% of thetotal milk volume and therefore was not expected to influence pHdynamics.

Testing total microbial and fungal agents: This was tested on Saburoagar using the “sedimentation” method. Uncovered plates with Saburo agarwere placed for 8 hours in the open. A piece of Nafion (10 mm×10 mm) wasplaced on the testing plate with active side down. Following overnightincubation at 37° C., the number of colonies was evaluated. Test andcontrol groups were compared.

Results

The pH results of the milk following test 1 are recorded in Table 12hereinbelow.

TABLE 12 1 day 2 day 3 day 4 day 5 day 6 day Film 1 7.4 7.2 6.9 6.8 6.76.3 Film 2 7.4 7.3 6.8 6.6 6.2 6.1 Film 3 7.4 7.3 6.9 5.9 5.4 4.9 Film 47.4 7.0 6.6 6.1 5.5 4.7 Film 5 7.4 6.8 6.2 5.6 4.4 3.7 Film 6. 7.4 7.06.6 5.6 4.8 4.1 Control 7.4 6.9 6.1 5.4 4.1 4.0

The pH results of the milk (test 1, repeat experiment) are recorded inTable 13 hereinbelow.

TABLE 13 Day pH Day 0 8.5 Day 1 8.7 Day 2 8.8 Day 3 8.7 Day 4 8.5 Day 58.6 Day 6 8.9 Day 7 8.5 Day 8 8.3 Day 9 8.5 Day 10 8.7 Day 11 8.8 Day 128.5 Day 13 8.5 Day 14 8.4 Day 15 8.5

The pH results of the 14 day test (test 2) are recorded in Table 14hereinbelow.

TABLE 14 1 day 2 day 3 day 4 day 5 day 6 day 7 day Nafion 1 7.5 7.4 7.37.1 7.1 7 6.8 Nafion 2 6.8 6.6 6.6 6.7 6.6 6.5 6.5 Control 7.4 6.7 6.25.1 4.2 4.1 4.1 8 day 9 day 10 day 11 day 12 day 13 day 14 day Nafion 16.8 6.6 6.6 6.7 6.6 6.5 6.5 Nafion 2 4.7 4.6 4.4 4.5 4.4 4.4 4.3 Control4.2 4.2 4.1 4.1 4.2 4.1 4.1

The results from testing total microbial and fungal agents are recordedin Table 15 hereinbelow.

TABLE 15 Total microbial and fungi agents (colonies) No First SecondControl (colonies) 1 2 1 14 2 2 2 31 3 3 3 24 4 0 6 25 5 4 5 16 6 0 2 207 2 5 19 8 3 2 13 9 2 2 37 10 1 3 25

Example 14 Cytotoxicity Testing of Second Series of Laminates

A second series of polyester base laminates were prepared bythermoplastic lamination methods. The laminates consisted of activeanionic components in a PVOH matrix. In some samples the active layerwas over-coated with a layer of PVOH.

The compositions and structure of laminates are provided in Table 16hereinbelow,

TABLE 16 Coating Coating Formulation (PVOH + Active Component) T(gsm)Ratio T₁(μ) T₂(μ) 1 PVOH + p Toluene sulphonic acid salt of Poly 0.9 3/218 0 (Dimethylamineethylmethacrylate) 2 PVOH + p Toluene sulphonic acidsalt of Poly 1.8 3/2 9 0 (Dimethylamineethylmethacrylate) 3 PVOH + pToluene sulphonic acid salt of Poly 0.9 3/2 9 24(Dimethylamineethylmethacrylate) 4 PVOH + p Toluene sulphonic acid saltof Poly 0.9 3/2 9 100 (Dimethylamineethylmethacrylate) 5 PVOH + pToluene sulphonic acid salt of Poly 0.9 3/2 9 0(Dimethylamineethylmethacrylate) 6 PVOH + p Toluene sulphonic acid saltof Poly 1.8 3/2 18 0 (Dimethylamineethylmethacrylate) 7 PVOH + p Toluenesulphonic acid salt of Poly 0.9 3/2 9 24(Dimethylamineethylmethacrylate) 8 As above + Laponite 0.58 4/1 6 0(T—total thickness in gram/square meter; R—ratio of the active componentand the PVOH binder; T₁—approximate thickness in microns, T₂—thicknessin microns of the overlay PVOH layer).

Materials and Methods

Determination of pH: Following wetting of the films in water, pH wasdetermined using pH-Fix 0-14 (Macherey-Nagel).

Cytotoxicity testing: Cytotoxicity tests were performed as described inExamples 12 and 13.

Results

The pH results are set forth in Table 17 hereinbelow.

TABLE 17 Film pH 1. MVC/HT/58/AY 5.0 2. MVC/HT/58/AY - pH 5.0 5.0 3.MVC/HT/58/AYTCG - pH 6.0 6.0 4. MVC/HT/58/AYTCG - pH 6.0 6.0 5.MVC/HT/58/BY <5.0 (4.8) 6. MVC/HT/58/BR 4.0 7. MVC/HT/58/BYTCG <5.0(4.8) 8. MVC/HT/58/CY 6.0

The cytotoxicity results are set forth in Table 18 hereinbelow.Cytotoxic effect was measured as the % of PI-stained (dead) cells.Following 20 minutes, approximately 80% of cells were green in Controlsample (without film).

TABLE 18 Cytotoxic effect, % No. Designation 1 min 2 min 10 min 20 min 1AY 95 ND 100 ND 2 AR 85 ND 100 ND 3 AYTCG 95 ND 100 ND 4 AYTCB  50* 90100 ND 5 BY 90 ND 100 ND 6 BR  70* ND  100* ND 7 BYTCG  5 ND  100* 100 8CY  10* ND  50*  50*

Of note, samples 3, 4 and 7 have a neutral PVOH overcoat and stilldemonstrated high cytotoxicity.

Example 15 Antibacterial Fabric

In order to ascertain whether the materials of the present invention canbe used to prevent necrosis, ion exchange resin beads were incorporatedin cotton fabric and contacted with a necrotic tissue for two days.

Material and Methods

Initial material: Biorad ion exchange resin AG 501-X8 (D) (catalogN142-6425). Approximately 100 beads were added per cm² of cotton fabric.

Results

As illustrated in FIGS. 9A-C, cotton comprising biorad ion exchangeresin had anti-necrotic effects.

Conclusion

Materials comprising ion exchange resins having buffer properties areeffective at preventing necrosis.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A method of generating a change in a cellular process of a targetcell of a multicellular organism resulting in death of said target cell,said method comprising contacting said target cell with a solid buffercomprising a sulfonated tetrafluoroethylene copolymer so as to alter anintracellular pH value in at least a portion of said target cell,thereby generating said change in a cellular process of a target cell ofa multicellular organism.
 2. The method of claim 1, wherein said targetcell is prokaryotic cell.
 3. The method of claim 1, wherein saidcontacting is effected ex vivo.
 4. The method of claim 1, wherein saidsolid buffer is attached to an affinity moiety.
 5. The method of claim1, wherein said solid buffer is at least partially covered by aselective barrier.
 6. The method of claim 1, wherein said solid buffercomprises a buffering layer and a water permeable layer being disposedon an external surface of said buffering layer.
 7. The method of claim1, wherein said solid buffer has a volumetric buffering capacity greaterthan about 20 mM H⁺/ml/pH.
 8. The method of claim 1, wherein said solidbuffer has a pH greater than pH
 8. 9. The method of claim 1, whereinsaid solid buffer has a pH less than pH 4.5.
 10. The method of claim 1,wherein said solid buffer is attached to at least part of a surface of asupport.
 11. A method of generating a change in a cellular process of atarget cell resulting in death of said target cell, said methodcomprising contacting the target cell with a solid buffer comprising asulfonated tetrafluoroethylene copolymer, wherein said solid buffercomprises a buffering layer and a water permeable layer being disposedon an external surface of said buffering layer, so as to alter anintracellular pH value in at least a portion of said target cell,thereby killing said target cell.
 12. The method of claim 11, whereinsaid target cell is a prokaryotic cell.
 13. The method of claim 11,wherein said contacting is effected ex vivo.
 14. The method of claim 11,wherein said solid buffer is internalized by said target cell.
 15. Themethod of claim 11, wherein said solid buffer is attached to an affinitymoiety.
 16. The method of claim 11, wherein said solid buffer is atleast partially covered by a selective barrier.
 17. The method of claim11, wherein said solid buffer has a volumetric buffering capacitygreater than about 20 mM H⁺/ml/pH.
 18. The method of claim 11, whereinsaid solid buffer has a pH greater than pH
 8. 19. The method of claim11, wherein said solid buffer has a pH less than pH 4.5.
 20. The methodof claim 11, wherein said buffer is attached to at least part of asupport.